Many glycoproteins having sugar chains have an important function in living organisms. In addition, it is elucidated that removing a sugar chain from glycoprotein leads to disappearance of biological activity about erythropoietin (EPO) and tissue plasminogen activator (TPA) etc. (Yo Kibata, “Protein Nucleic Acids Enzyme” Vol. 36, p 775 (1991); Makoto Takeuchi “Biochemistry” Vol. 62, p 1272 (1990)). This shows that a sugar chain in glycoprotein plays an important role in biological activity. Therefore, producing glycoprotein efficiently is preferable in developing medicines.
In the case of producing glycoprotein, using yeast is considered from a viewpoint of applying techniques of genetics and molecular biology, or from a viewpoint of being a single eukaryote which has high productivity of foreign protein. On the other hand, a sugar chain produced by yeast, having many mannose added structure, shows high antigenicity in a body of higher animals. For this reason, there is a problem that glycoprotein produced by yeast might not be adequate for drugs especially being administrated through blood vessels. To resolve this problem, an yeast strain which produces a sugar chain without antigenicity was established by gene-disruption (for example, see Japanese publication of patent application 1994-277086 bulletin, Japanese publication of patent application 1995-299509 bulletin and Japanese publication of patent application 2001-161376 bulletin.)
Especially triple disruptant comprising och1 disruption (Δoch1), mnn1 disruption (Δmnn1) and mnn4 disruption (Δmnn4) is disclosed in international publication WO01/014522 pamphlet (see below patent literature 1). The document discloses the yeast any of the following gene, which relates to production of an outer sugar chain and yeast specific, is destroyed or any mutation is introduced to the following genes: alpha-1, 6 mannosyl transferase conducting first elongation reaction (OCH1); a gene coding for alpha-1, 3 mannosyl transferase which adds mannose to nonreducing end of sugar chain (MNN1); and a gene regulating addition of mannose-1-phosphate (MNN4). The yeast strain is thought to be useful for developing functional food and drugs because it is excellent in production of glycoprotein with mammalian type sugar chain.
However, the gene-disruptant shows higher temperature sensitivity than a wild strain and lower growth activity, for example, it doesn't grow at 37 degrees C. As a result, compared to wild strains, a gene-disruptant shows defective growth and has a problem that protein productivity is low.
Beta-glucan, which is a kind of polysaccharide, is known to have effects of activating macrophages, NK cells, T cells and killer T cells that attack infected cells and cancer cells in the body, and increasing immunity and resistance. With this immunity increasing effect, ability to exclude bacteria and foreign substances having entered the body is increased, so that resistance for inhibiting development of disease even if infected can be obtained. Also, with such an increase in immunity, effects of reducing allergic reactions and suppressing tumor such as cancer can be expected and antitumor effect has been actually revealed by various clinical tests. Moreover, effects such as a decrease in blood glucose level, diuretic effect, blood pressure adjustment, decrease in blood cholesterol and neutral fat levels can also be obtained.
Yeasts (especially baker's yeast) have long been used for fermented food and are extremely safe as food products. The baker's yeast, usually including about 45% of beta-glucan in the cell wall, is commercialized as a dietary supplement with a target narrowed down to immunity increasing effect. The beta-glucan of the baker's yeast is utilized by being extracted mainly from the cell wall. The beta-glucan derived from the baker's yeast is sold in the United States mainly as zymosan.
In order to obtain more beta-glucan from the cell wall of yeast, culturing yeast in large scale is required. Also, while a high-efficient extraction of beta-glucan is required after the culture, this operation is not easy since a specific technique is required. Therefore, development of a method capable of producing beta-glucan derived from yeast more easily and inexpensively by skipping such processes as much as possible is desired.
On the other hand, a mutagenesis method inducing mutation by making more than two kinds of DNA polymerase with different fidelity coexist in single Escherichia coli cell is known.
Moreover, in the international publication WO00/028015 pamphlet (following patent literature 2), “A method of mutation induction to a gene characterized by introducing more point mutation into one strand than the other strand of double stranded genome DNA of a cell or a living organism” (claim 1 of the bulletin) is disclosed. In the bulletin, “Mutants can be obtained effectively by accumulating more random point mutations into one DNA strand than the other strand and by reducing risks for extinction of mutagenized cells of living organisms while increasing mutation rate” is described (3rd line from the bottom of page 9 onward in the bulletin). However, while an example using Escherichia coli is found in the bulletin, an example using yeast is not found. Therefore, it is not quite clear about what kind of mutation is induced in the case of applying the technique disclosed in the bulletin to yeast.
(Patent literature 1) International publication WO01/014522 pamphlet
(Patent literature 2) International publication WO00/028015 pamphlet